Des-2-serine, 3 asparagine-calcitonin

ABSTRACT

New peptides are disclosed which have biological activity of the same type as known calcitonins and which do not have a serine in position 2 nor an asparagine in position 3. Both serine 2 and asparagine 3 are omitted. Also resin peptides are disclosed which may be converted to peptides having such biological activity; and processes for producing said resin peptides and said calcitonin peptides.

FIELD OF THE INVENTION

This invention relates to calcitonins having biological activity and topeptides which can be converted to biologically active calcitonins andto processes for preparing such peptides and calcitonins.

BACKGROUND OF THE INVENTION

All known natural calcitonin peptides contain an amino acid sequence of32 amino acids. The natural calcitonins include the salmon, eel, bovine,porcine, ovine, rat and human calcitonins. Salmon calcitonin, forexample, has the following formula: ##STR1##

In U.S. Pat. Nos. 3,926,938, 4,062,815, 3,929,758, 4,033,940, 4,336,187,4,388,235 and 4,391,747 are disclosed improved syntheses of calcitoninsincluding the salmon calcitonin referred to above.

SUMMARY OF THE INVENTION

We have discovered synthetic calcitonin analogs of the above-mentionednaturally occurring calcitonin peptides with 30 amino acids that havebiological activity of the same type as the known calcitonins. Asignificant difference in structure is that in our new peptides serine 2and asparagine 3 are omitted from the sequence. These new peptides aretermed des-2-serine, 3-asparagine-calcitonin, using the IUPAC-IUB methodof nomenclature for synthetic modifications of peptides [Biochem.Biophys. Acta, 133, 1-5 (1967)]. The new peptides have higher potencyand quality when compared to known calcitonins.

DESCRIPTION OF INVENTION

The formula of our new des-2-serine, 3-asparagine-salmon calcitoninhaving activity of the same type as known salmon calcitonin may bewritten as follows: ##STR2##

The formula of our new des-2-serine, 3-asparagine-eel calcitonin may bewritten as follows: ##STR3##

Monikawa et al. [Experientia, 32(9), 1104-1106 (1976)] showed thatsynthetic eel calcitonin has a hypocalcemic potency around 4300 IU/mg.,while the synthetic analog (1,7-α-L-amino-suberic acid)eel calcitoninhas a hypocalcemic potency of about 3400 IU/mg.

Therefore, we also include within the present invention the30-amino-acid analogs of salmon and eel calcitonin in which thecysteines in the first and seventh positions are replaced byα-L-aminosuberic acid and serine 2 and asparagine 3 are omitted from thesequence.

As may be seen from the formula given above, 30 amino acids are involvedand in this formula the positions are numbered according to the acceptedprocedure beginning at position 1 for the Cys on one end of the chain,and ending with Pro at position 30 at the other end of the chain. Forclarity of description, this same numbering system will be followed inreferring to the cycles of the synthesis. The assembly of the aminoacids begins with cycle 30 which involves the coupling of proline andcontinues with cycle 29 which involves the coupling of threonine, etc.

In general we use a solid phase methodology of synthesis and start witha resin called benzhydrylamine resin (BHA resin). This resin is derivedfrom a cross-linked polystyrene bead resin manufactured bycopolymerization of styrene and divinylbenzene. Resin of this type isknown and its preparation is further demonstrated by Pietta et al.[Pietta, P. S. and Marshall, G. R., Chem. Commun., 650 (1970)], andOrlowski et al., [J. Org. Chem., 41, 3701 (1976)]. The cross-linkedpolystyrene BHA resin is available from chemical supply houses. We usethe designation ##STR4## to represent the BHA resin in which ○P is thepolystyrene portion of the resin.

RESIN PEPTIDE SYNTHESIS

In this synthesis the amino acids are added one at a time to theinsoluble resin until the total peptide sequence has been built up onthe resin. The functional groups of the amino acids are protected byblocking groups. The α-amino group of the amino acids is protected by atertiary butyloxycarbonyl group or an equivalent thereof. Thisα-tertiary butyloxycarbonyl group we designate as BOC. The hydroxylfunctions of serine and threonine are protected by a benzyl or benzylderivative group such as 4-methoxybenzyl, 4-methylbenzyl,3,4-dimethylbenzyl, 4-chlorobenzyl, 2,6-dichlorobenzyl, 4-nitrobenzyl,benzhydryl or an equivalent thereof. We use the term Bz to represent thebenzyl or benzyl derivative group.

The hydroxyl function of tyrosine may be unprotected, may be protectedby a benzyl or benzyl derivative group as described above, as a Bzgroup, or may be protected by a benzyloxycarbonyl or a benzyloxycarbonylderivative such as 2-chlorobenzyloxycarbonyl or 2-bromobenzyloxycarbonylgroup or equivalent thereof. We use the term W to represent either noprotective group, a Bz group, a benzyloxycarbonyl group or abenzyloxycarbonyl derivative group.

The thiol function of cysteine may be protected by benzyl or benzylderivative protective groups described above and designated Bz, or by ann-alkylthio group such as methylthio, ethylthio, n-propylthio,n-butylthio or equivalents thereof. We use the character R₂ to representan n-alkylthio group or Bz, and the character R₁ to represent Bz when R₂is n-alkylthio and to represent n-alkylthio when R₂ is Bz.Alternatively, R₁ may be another cysteine group and when this is thecase R₂ is Bz. The guanidine function of arginine may be protected by anitro group, a tosyl group or an equivalent thereof. We use thecharacter T to represent either a nitro group or a tosyl group. Theε-amino function of lysine may be protected by a benzyloxycarbonyl groupor a benzyloxycarbonyl derivative such as a 2-chlorobenzyloxycarbonyl,3,4-diethylbenzyloxycarbonyl, or equivalents thereof. We use thecharacter V to represent benzyloxycarbonyl group or a benzyloxycarbonylderivative group. The protective groups used on the imidazole nitrogenof histidine are the benzyloxycarbonyl group and benzyloxycarbonylderivatives such as described above for lysine and are designated as V.The γ-carboxylic acid group of glutamic acid is protected by a benzyl orbenzyl derivative group such as described for the protection hydroxylfunction of serine and threonine. These protective groups arerepresented by the character Bz.

Preferred amino acid reactants for use in each of the 30 cycles of thesynthesis of salmon calcitonin (used for exemplification only) are givenin the following Table I:

                  TABLE I                                                         ______________________________________                                        Cycle                                                                         Number Amino Acid Reactant                                                    ______________________________________                                        30     BOC-L-proline                                                          29     BOC-O--benzyl-L-threonine                                              28     BOC-glycine                                                            27     BOC-O--benzyl-L-serine                                                 26     BOC-glycine                                                            25     BOC-O--benzyl-L-threonine                                              24     BOC-L-asparagine p-nitrophenyl ester                                   23     BOC-O--benzyl-L-threonine                                              22     BOC-ω-nitro-L-arginine or BOC-ω-tosyl-                            L-arginine                                                             21     BOC-L-proline                                                          20     BOC-O--bromobenzyloxycarbonyl-L-tyrosine                               19     BOC-2-O--benzyl-L-threonine                                            18     BOC-L-glutamine p-nitrophenyl ester                                    17     BOC-L-leucine                                                          16     BOC-ε-CBZ-L-lysine or BOC-ε-2-chlorobenzyloxy-                carbonyl-L-lysine                                                      15     BOC-N(im)-CBZ-L-histidine                                              14     BOC-L-leucine                                                          13     BOC-L-glutamic acid γ-benzyl ester                               12     BOC-L-glutamine p-nitrophenyl ester                                    11     BOC-O--benzyl-L-serine                                                 10     BOC-L-leucine                                                           9     BOC-ε-CBZ-L-lysine or BOC-ε-2-chlorobenzyloxy-                carbonyl-L-lysine                                                       8     BOC-glycine                                                             7     BOC-L-leucine                                                           6     BOC-L-valine                                                            5     BOC-S--ethylthio-L-cysteine, BOC-S--methylthio-L-                             cysteine, BOC-S--n-propylthio-L-cysteine or                                   BOC-S--n-butylthio-L-cysteine                                           4     BOC-O--benzyl-L-threonine                                               3     BOC-O--benzyl-L-serine                                                  2     BOC-L-leucine                                                           1     BOC-S--p-methoxybenzyl-L-cysteine, BOC-S--3,4-                                dimethylbenzyl-L-cysteine or BIS-BOC-L-cysteine.                       ______________________________________                                    

Each of the amino acid derivatives mentioned in Table I may be purchasedfrom supply houses.

CYCLE 30 Coupling Of Proline To BHA Resin

The reaction vessel used in all steps of the resin peptide synthesis maybe a glass vessel equipped with inlet ports at the top for addition ofmaterials and a sintered glass disk at the bottom for removal of solublereaction mixtures and wash solvents by filtration. Filtration may beperformed either by vacuum or the use of nitrogen pressure. The contentsof the vessel may be agitated by shaking the entire vessel or bymechanical stirrer.

In cycle 30 the BHA resin is placed in the reaction vessel and suspendedin a solvent such as methylene chloride, chloroform, dimethylformamide,benzene or equivalents thereof in proportions of about 3 to 12 ml. ofsolvent per gram of resin. To this is added BOC-L-proline in an amountof about 1 to 6 equivalents per free amine equivalent of the BHA resinemployed. After a period of mixing of 5 to 10 minutes, a couplingreagent (CA) such as dicyclohexylcarbodiimide (DCC) may be added, orother diimide coupling agents may be used. The diimide coupling agentmay be used in the amount of 0.5 to 2.0 equivalents per equivalent ofBOC-L-proline used.

The BOC-L-proline may be coupled in the absence of a coupling reagent ifits active ester derivative, its azide derivative, its symmetricalanhydride derivative, or a suitably chosen mixed anhydride derivative isused. Active ester derivatives that may be employed are 2-nitrophenylester, 4-nitrophenyl ester, pentafluorophenyl ester, N-hydroxysuccimideester or equivalents thereof. The active esters are used in amounts of 1to 10 equivalents per free amine equivalent of BHA resin.

The reaction mixture consisting of the BHA resin, the solvent, theBOC-L-proline, and the coupling reagent or BOC-L-proline active ester isstirred or shaken mechanically until the reaction is complete asindicated by a ninhydrin test [E. Kaiser, et al., Anal. Biochem., 34,595-8 (1970)] on a test sample. After completion of the couplingreaction, the BOC-L-proline resin may be washed with solvents such asmethylene chloride, chloroform, methyl alcohol, benzene,dimethylformamide, or acetic acid. The amount of wash solvent maysuitably be 5 to 20 ml. of solvent for each gram of BHA resin usedinitially. If it is desired to terminate the coupling reaction beforecompletion, the washing procedure may be used and the remaining freeamino groups on the BOC-L-proline resin may be blocked from furtherreaction by acetylation with an excess of acetylation reagents. Theacetylation procedure may be performed by agitating the BOC-L-prolineresin with a solution of the acetylation reagent for a period of 0.5 to12 hours. Acetylation reagents such as N-acetylimidazole in methylenechloride solution or a mixture of acetic anhydride and triethylamine inchloroform may be used. The acetylation reagent may be used in theamount of 0.5 to 5.0 equivalents per equivalent of free amine titer ofthe starting BHA resin.

The coupling reaction to produce the BOC-L-proline resin may bedescribed by the following formula: ##STR5##

Deprotection of BOC-L-Proline Resin

The BOC-L-proline resin produced as above described may be washed with asolvent such as referred to above and deprotected by agitating it withan agent such as a mixture of trifluoroacetic acid (TFA) in a solventsuch as methylene chloride, chloroform, benzene or equivalents thereof.The amount of TFA in the solvent may vary from 10 to 100% of themixture. The amount of TFA-solvent mixture may vary from 3 to 20 ml. pergram of BHA resin used initially. The reaction time may be from about 10minutes to 4 hours. The deprotection step is terminated by filtration toremove the TFA-solvent mixture. The residual TFA may be removed from theL-proline resin by washing with 3 to 20 ml. per gram of BHA resin of a 5to 30% of triethylamine solution in a solvent such as methylenechloride, chloroform, benzene or equivalents thereof. Other tertiary orsecondary organic amines may be used in place of the triethylamine, suchas, trimethylamine, N-ethylpiperidine, diisopropylamine or equivalentsthereof. The free amine titer of the L-proline resin may be determinedby the Dorman titration procedure [Dorman, L. C., Tetrahedron Letters,1969, 2319-21]. The deprotection reaction may be described by thefollowing formula: ##STR6##

CYCLE 29

The prolyl-BHA resin obtained as a result of cycle 30 may be suspendedin a coupling solvent, BOC-O-Bz-L-threonine added and the mixtureequilibrated in the same manner. The coupling agent, DCC, may be added,and after completion of the reaction as indicated by the isatin test [E.Kaiser, et al., Anal. Chem. Acta, 118, 149-51 (1980)], the reactionmixture may be removed from the BOC-O-Bz-threonylprolyl-BHA resin byfiltration. The peptide resin may be washed with solvents. The amountsof reactants and solvents and reaction times may be the same asdescribed in cycle 30. The BOC group may be removed from the peptideresin by the deprotection method described in the cycle 30. Theresulting O-Bz-threonylprolyl-BHA resin is then ready for cycle 28. Thereactions of the cycle 30 may be shown by the following formula:##STR7##

For convenience, we may write this resulting resin peptide usingabbreviated nomenclature as follows: ##STR8##

CYCLE 28

In cycle 28, the coupling reaction and also the deprotection reactionmay be performed in the same manner as in cycle 29 except thatBOC-glycine is used in place of BOC-O-Bz-L-threonine. The reactionthrough coupling and deprotection may be written: ##STR9##

CYCLE 27

In cycle 27, the coupling and deprotection reactions may be performed inthe same manner as in cycle 29 except for the substitution ofBOC-O-Bz-L-serine as the amino acid derivative. This may be written:##STR10##

CYCLE 26

In cycle 26, the coupling and deprotection reactions are performed asdescribed in cycle 29 except that BOC-glycine is substituted as theamino acid reactant. These reactions through coupling and deprotectionmay be written as follows: ##STR11##

CYCLE 25

In this cycle, the coupling and deprotection reactions may be as incycle 29 using the same amino acid reactant, resulting in the followingcompound: ##STR12##

CYCLE 24

In cycle 24, the coupling reaction is performed using an active esterderivative of BOC-L-asparagine. The active ester procedure is used inplace of the DCC coupling agent with BOC-L-asparagine orBOC-L-glutamine. The reaction using the active ester derivative ofBOC-L-asparagine may be performed in the amount of 2 to 10 equivalentsper free amine equivalent of BHA resin in dimethylformamide, mixtures ofdimethylformamide with benzene, methylene chloride or chloroform or withequivalents thereof in the amount of 2 to 20 ml. of solvent per gram ofBHA resin used initially.

Reaction times range from 1 to 72 hours. The reaction mixture may beremoved from the BOC peptide resin by filtration after completion of thereaction as indicated by a ninhydrin test. The active ester derivativesemployed may be 2-nitrophenyl esters, 4-nitrophenyl esters,pentafluorophenyl esters, or equivalents thereof. We use AE to designatethe active ester portion of the derivative. The coupling reaction may bewritten: ##STR13##

The deprotection reaction to remove the BOC group is performed as incycle 30.

CYCLES 23-19

In each of cycles 23 to 19, the coupling and deprotection reactions maybe conducted using the methods and proportions of reactants as in cycle29, using BOC-O-Bz-L-threonine in cycle 23, BOC-ω-T-L-arginine in cycle22, BOC-L-proline in cycle 21, BOC-L-L-tyrosine in cycle 20, andBOC-O-Bz-L-threonine in cycle 19. The compound resulting from thecompletion of cycle 19 may be written: ##STR14##

CYCLE 18

In cycle 18, the coupling and deprotection reactions may be performedusing the methods and proportions of reactants as in cycle 24 using aBOC-L-glutamine active ester derivative as the amino acid derivative,resulting in the compound: ##STR15##

CYCLE 17

In cycle 17, the reactions are performed as in cycle 28 usingBOC-L-leucine as the amino acid derivative. The compound resulting fromcycle 17 is: ##STR16##

CYCLE 16

In cycle 16, we may use as the amino acid derivative BOC-ε-V-L-lysine.Otherwise, cycle 17 methods may be performed as in cycle 29 resulting inthe compound: ##STR17##

CYCLES 15-13

Cycles 15 to 13 may be performed as in cycle 29 except for the use ofBOC-N(im)-V-L-histidine in cycle 15, BOC-L-leucine as the reactant incycle 14 and BOC-L-glutamic acid Bz ester (Bz represents the same groupsas it represents for serine and threonine) as the reactant in cycle 13,resulting in the following compound from cycle 13: ##STR18##

CYCLES 12-6

Cycle 12 may be performed identically to cycle 18 usingBOC-L-glutamine-AE as the amino acid derivative. Cycles 11 to 16 may beperformed as in cycle 29 except for the use of BOC-O-Bz-L-serine incycle 11, BOC-L-leucine in cycle 10, BOC-ε-V-L-lysine in cycle 9,BOC-glycine in cycle 8, BOC-L-leucine in cycle 7, and BOC-L-valine incycle 6 resulting in the compound: ##STR19##

CYCLE 5

Cycle 5 may be performed as in cycle 29 except for the use ofBOC-S-ethylthio-L-cysteine or equivalent for the amino acid derivative.The compound resulting from cycle 5 is described by the formula:##STR20## wherein R₂ is an alkylthio or a Bz group.

CYCLES 4-2

Cycle 4 may be performed as in cycle 29 except that BOC-O-Bz-L-threoninebe used as the amino acid derivative in cycle 4, and BOC-O-Bz-L-serinemay be used as the amino acid derivative in cycle 3 and cycle 2 may beperformed identically to cycle 17. The compound resulting from cycle 2is: ##STR21##

CYCLE 1

This cycle may be performed identically to cycle 5 using BOC-S-R₁-L-cysteine derivatives. The R₁ group chosen for the cysteine may be thesame as used in cycle 5 or different. For example, if the derivativechosen for cycle 5 is BOC-S-ethylthio-L-cysteine, the derivative incycle 5 may be BOC-S-4-methoxybenzyl-L-cysteine or ifBOC-S-4-methoxybenzyl-L-cysteine was chosen for cycle 5, then thisderivative or BOC-S-ethylthio-L-cysteine may also be used in cycle 1.The compounds resulting from cycle 1 are illustrated by the formula:##STR22## where R₁ is S-n-alkyl, Cys or Bz and R₂ is S-n-alkyl or Bz; R₁being S-n-alkyl or Cys when R₂ is Bz, and R₁ being Bz when R₂ isS-n-alkyl.

Cycle 1 represents the completion of the resin peptide. The resinpeptide may be removed from the reaction vessel and dried in a vacuum.The weight of the resin peptide may be expected to be from 2.0 to 3.5times the weight of BHA resin used initially in the synthesis.

Resin Peptide Cleavage

The peptide is cleaved from the resin peptide resulting from cycle 1 bytreatment with liquid hydrogen fluoride (HF). The HF cleavage reactionmay be performed by treating a mixture of the resin peptide and anisole(0.5 to 5 ml. for each gram of resin peptide) with liquid HF (2 to 20ml. for each gram of resin peptide) for 0.5 to 20 hours at -20 degreesof +15 degrees centigrade. After the reaction period, the excess HF maybe removed by evaporation and the resulting mixture of peptide and resinbeads may be extracted with organic solvent such as ethyl acetate,diethyl ether, benzene or the like to remove the anisole and residualHF. The peptide may be separated from the resin beads by extraction intoaqueous acetic acid. The peptide at this stage is not cyclic but is thenon-cyclic product without the disulfide bond between the cysteines atpositions 1 and 5 in the molecule.

The HF treatment removes all blocking groups from the peptide, exceptthe S-alkylthio blocking groups on the thiol function of cysteineresidue at either position 1 or 5. The S-n-alkylthio-L-cysteine residueis stable to the HF cleavage procedure and remains intact throughout thecleavage and extracting procedures. The S-Bz-L-cysteine residue iscleaved by HF to yield a cysteine residue with a free thiol function.Both types of blocking groups have been employed during our synthesis incombination with each other at positions 1 and 5.

Thus, the peptides obtained after HF cleavage can be one of four typesdepending upon the blocking groups chosen for the thiol function of thecysteine derivative used during the resin peptide synthesis.

If BOC-S-Bz-L-cysteine derivatives are used in the resin peptidesynthesis cycle 1 and BOC-S-n-alkylthio-L-cysteine is used in cycle 5,the peptide resulting after HF cleavage would be of Type I and wouldhave a free thiol function at position 1 and have a S-n-alkylthiofunction on the cysteine residue at position 5. We call this a Type Ipeptide which is represented by the formula given as follows: ##STR23##

Conversely, if BOC-S-n-alkylthio-L-cysteine derivative is used in cycle1 and the BOC-S-Bz-L-cysteines were used in position 5, the peptideresulting from the cleavage would be of Type II and would be representedby the formula: ##STR24##

In place of the protecting group S-n-alkyl at position 1 we may use aS-cysteinyl group (which with the cysteine at this position forms acysteine group) and when we do this we use a Bz group for protecting thecysteine at position 5. If Bis-BOC-L-cystein is used as the reactant incycle 1 and the BOC-S-Bz-L-cysteine is used as the reactant in cycle 5,the peptide resulting from the cleavage will be of Type III and may berepresented by the following formula: ##STR25##

If BOC-S-Bz-L-cysteine is used as the reactant in both positions 1 and5, the peptide resulting from the cleavage will be of Type IV andrepresented by the following formula:

Cys-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH₂.

The conversion of Types I, II and III peptides to the cyclic disulfidepeptides may be performed by diluting with distilled water the aqueousacetic acid solution of the crude peptides from HF cleavage to a finalvolume of 50 to 200 ml. per gram of resin peptide cleaved. The pH ofthis solution may be adjusted from 5 to 10 by the addition of ammoniumhydroxide solution and the mixture may be stirred in a closed containerunder a stream of an inert gas such as nitrogen for about 2 to 48 hours.The reaction period can be stopped when the effluent gas stream nolonger contains n-alkylmercaptan. The pH of the reaction mixture may belowered to about 3.5 to 5.5 by the addition of glacial acetic acid.

The conversion to Type IV peptides to the cyclic disulfide peptide maybe performed by the classical method known to the art in which thepeptides are oxidized to form a ring structure to include the cysteinesat positions 1 and 5.

Whether the intermediate peptides are of Type I, II, III or IV we maysynthesize peptides having amino acid chains corresponding to any knowncalcitonin. Such peptide synthesized as herein set forth, may bepurified and found to have the same type of biological activity as theknown calcitonin. Any calcitonin so synthesized is designateddes-2-serine, 3-asparagine-calcitonin. This is in accordance with theIUPAC-IUB method of nomenclature.

Purification Of The Crude des-2-serine, 3-asparagine-Salmon Calcitonin

The crude peptide solutions at pH 5.0 from the above synthesis may beconcentrated using an ion-exchange procedure. The concentrate may bepurified by a combination of gel-filtration procedures, ion-exchangechromatography methods and partition chromatography. The final purifiedproduct may be obtained from solution by freeze-drying as a fluffy whitesolid. The product gives the correct amino acid analysis for the desiredpeptide.

Following is the specific example of the preparation of the peptide.

EXAMPLE 1 Resin Activation

The BHA resin (5 g.) with an amine titer of 0.61 meq./g. was placed inthe reacter vessel of a peptide synthesizer marketed by VegaBiochemicals of Tucson, Ariz. The resin was treated with 25 ml. of thefollowing solvents filtering after each treatment:

Methylene chloride for 2 minutes

Chloroform for 2 minutes two times each

10% triethylamine in chloroform for 5 minutes two times each

Chloroform for 2 minutes

Methylene chloride for 2 minutes three times each.

Cycle 30

Coupling:

The BHA resin, 25 ml. of methylene chloride and 1.31 g. (0.0061 moles)of BOC-L-proline was stirred for 10 minutes. 6.1 ml. of methylenechloride solution of dicyclohexylcarbodiimide (1 milliequivalent of DCCper 1 ml. of solution) was added to the reactor and the mixture agitatedfor 6 hours. The reaction mixture was removed from the reactor byfiltration and the BOC-prolyl BHA resin was subjected to the followingsuccessive 2 minute, 25 ml. washes, removing the wash by filtration eachtime:

Methylene chloride two times,

Methyl alcohol two times,

Methylene chloride three times.

Acetylation:

The resin was then agitated with a mixture of 1.5 ml. of triethylamine(TEA), 1 ml. of acetic anhydride and 25 ml. of chloroform for 2 hours.The reaction mixture was removed by filtration and the resin subjectedto the following 2 minute, 25 ml. washes:

Chloroform two times

Methyl alcohol two times

Methylene chloride three times.

Deprotection:

The BOC-protected resin was agitated for 5 minutes with a mixture of 15ml. of trifluoroacetic acid (TFA) and 15 ml. of methylene chloride. Thismixture was removed by filtration and the resin was agitated with asecond mixture of 15 ml. of TFA and 15 ml. of methylene chloride for 30minutes. The reaction mixture was removed by filtration and the resinsubject to the following 25 ml. washes:

Methylene chloride two times two minutes each

Methyl alcohol two times two minutes each

Chloroform two times two minutes each

10% TEA in chloroform two times ten minutes each

Chloroform two times two minutes each

Methylene chloride two times two minutes each.

The L-proline BHA resin was titrated to establish the amine or prolinetiter. This value was 0.55 milliequivalents of amine or proline per gramof resin.

Cycle 29

Coupling:

The L-prolyl resin, 25 ml. of methylene chloride and 1.64 g. (0.0053mole) of BOC-O-benzyl-L-threonine were agitated for 10 minutes. Then 5.5ml. of methylene chloride solution of dicyclohexylcarbodiimide (1milliequivalent of DCC per 1 ml. of solution or a total of 0.0055 moleof DCC) was added to the reactor and the mixture agitated for 2 hours.The reaction mixture was removed from the reactor and the resin wassubjected to the following successive 2 minute, 25 ml. washes, removingthe wash by filtration each time.

Methylene chloride two times

Methyl alcohol two times

Methylene chloride three times

An isatin test was negative.

Deprotection:

The deprotection procedure described in cycle 30 was repeated for thiscycle.

Cycles 28 Through 25

The coupling and deprotection procedures used in these cycles were thesame as in cycle 30 except that the following amino acid derivativeswere used in place of the threonine derivative:

Cycle 28--0.93 g. (0.0053 mole) of BOC glycine

Cycle 27--1.55 g. (0.0053 mole) of BOC-O-benzyl-L-serine

Cycle 26--The reactant used was the same as cycle 28

Cycle 25--The reactant used was the same as cycle 29

Cycle 24

Coupling:

The peptide resin obtained from cycle 25 was washed twice with 25 ml.portions of dimethylformamide (DMF). The resin was then agitated for 24hours with a solution of 2.82 g. (0.008 mole) of BOC-L-asparaginep-nitrophenyl ester in 35 ml. of DMF. The reaction mixture was filteredand the resin peptide subjected to two minute washes with two successive25 ml. portions of the following solvents: DMF, methylene chloride,methanol, methylene chloride. Each individual solvent was removed byfiltration. A ninhydrin test was negative.

Deprotection:

The deprotection procedure used in cycle 30 was repeated.

Cycle 23

Coupling and deprotection procedures were the same as in cycle 29 usingthe same reactants and amounts.

Cycle 22

Coupling:

The resin peptide obtained from cycle 23 was washed with two successive25 ml. portions of DMF. The resin peptide was then agitated for 10minutes with a mixture of 3.42 g. (0.008 mole) ofBOC-N-ω-tosyl-L-arginine and 25 ml. of DMF. Then 8 ml. of DCC inmethylene chloride (equivalent to 0.008 mole of DCC) was added and themixture agitated for 6 hours. The reaction mixture was removed byfiltration. The resin peptide was subjected to two minute washes withtwo successive 25 ml. portions of the following solvents: DMF, methylenechloride, methyl alcohol, methylene chloride. The ninhydrin test wasnegative.

Deprotection:

The deprotection used in cycle 30 was repeated.

Cycle 21

Coupling:

The peptide resin obtained from cycle 22 was agitated for 10 minuteswith 1.72 g. (0.008 mole) of BOC-L-proline and 25 ml. of methylenechloride. 8 ml. of DCC in methylene chloride (equivalent to 0.008 moleof DCC) was added and the mixture agitated for 6 hours. The reactionmixture was removed by filtration and the resin peptide subjected to twominute washes with two successive 25 ml. portions of the followingsolvents: methylene chloride, methyl alcohol, methylene chloride. Eachindividual wash was removed by filtration. The ninhydrin test wasnegative.

Deprotection:

The deprotection used in cycle 30 was repeated.

Cycle 20 and 19

The coupling and deprotection procedures used in this cycle was the sameas in cycle 21 except that in the coupling reaction the following aminoacid derivative was used in place of BOC-L-proline.

Cycle 20--4.07 g. (0.008 mole) BOC-O-2-bromobenzyloxycarbonyl-L-tyrosine

Cycle 19--2.47 g. (0.008 mole) BOC-O-benzyl-L-threonine

Cycle 18

This procedure is the same as cycle 24 except that 2.94 g. (0.008 mole)of BOC-L-glutamine p-nitrophenyl ester is used in place of theasparagine derivative.

Cycles 17 through 13

The procedure is the same as used in cycle 29 except that the followingamino acid derivatives were used in place of the threonine derivative:

Cycle 17--1.32 g. (0.0053 mole) of BOC-L-leucine

Cycle 16--2.20 g. (0.0053 mole) of BOC-ε-2-chlorobenzyloxy-L-lysine

Cycle 15--2.06 g. (0.0053 mole) of BOC-N(im)-carbobenzyloxy-L-histidine

Cycle 14--1.32 g. (0.0053 mole) of BOC-L-leucine

Cycle 13--1.79 g. (0.0053 mole) of BOC-L-glutamic acid--γ-benzyl ester

Cycle 12

Same as cycle 18.

Cycle 11

The procedure used was the same as was used in cycle 21 except that inthe coupling reaction 2.36 g. (0.008 mole) of BOC-O-benzyl-L-serine wasused in place of the proline derivative.

Cycles 10 through 7

The procedures used were the same as used in cycle 29 except in thecoupling reactions the following amine acid derivatives were used inplace of the threonine derivative.

Cycle 10--Same reactants as used in cycle 17

Cycle 9--The reactants were the same as in cycle 16

Cycle 8--Same reactants as used in cycle 28

Cycle 7--Same reactants as used in cycle 17

Cycle 6

Coupling:

The resin peptide from cycle 7 was agitated for 10 minutes with 1.74 g.(0.008 mole) of BOC-L-valine and 25 ml. of methylene chloride. Then 8ml. of DCC in methylene chloride (equivalent to 0.008 mole of DCC) wasadded and the mixture agitated for 16 hours. The reaction mixture wasremoved by filtration. The resin peptide was subjected to two minutewashes with two successive 25 ml. portions of the following solvents:methylene chloride, methyl alcohol, methylene chloride. Each individualwash was removed by filtration.

Deprotection:

See cycle 29.

Cycle 5

The procedure was the same as used in cycle 29 except that in thecoupling reaction 1.59 g. (0.0053) of BOS-S-ethylthio-L-cysteine wasused in place of the threonine derivative.

Cycle 4

The reactants and procedures used were the same as cycle 29.

Cycle 3

The reactants and procedures used were the same as cycle 27.

Cycle 2

The reactants and procedures used were the same as cycle 17.

Cycle 1

The reactants and procedures used were the same as cycle 29 except that1.81 g. (0.0053 mole) of BOC-S-p-methoxybenzyl-L-cysteine was used inplace of the threonine derivative.

After completion of cycle 1, the resin peptide was washed with twosuccessive 25 ml. portions of n-hexane. The peptidic material wasremoved from the reactor and dried in an vacuum oven at 40 degreescentigrade and 0.1 mm. of Hg for 24 hours.

Cleavage with Hydrogen Fluoride

The dried resin peptide (2 g.) and 2 ml. of anisole were placed in aTeflon reaction vessel. The vessel equipped with a Teflon-coated magnetstirrer was placed in a dry ice-acetone bath and 15 ml. of hydrogenfluoride gas was condensed into the vessel. This mixture was stirred at0 degrees centigrade in an ice bath for 1 hour. The hydrogen fluoridewas removed by evaporation at reduced pressure. The residue wastriturated with six 25 ml. portions of ethyl acetate. The peptide wasextracted from the resin beads with 120 ml. of 0.1 molar aqueous aceticsolution.

Cyclization Of The Peptide

The aqueous acetic acid extract obtained from hydrogen fluoride cleavagewas diluted to 200 ml. by addition of 80 ml. of distilled water. The pHof the solution was adjusted to 7.5 by the addition of concentratedammonium hydroxide. The solution was stirred in a closed vessel under astream of nitrogen for 24 hours. At this time no ethyl mercaptan couldbe detected in the emerging nitrogen stream. The ethyl mercaptan contentof the nitrogen stream was measured by passing the stream through asolution of Ellman's reagent [Ellman, G. L., Arch. Biochem. Biophys.,82, 70-7 (1969)]. The pH of the reaction mixture was adjusted to 5.0 byaddition of glacial acetic acid.

Purification Of The Crude des-Ser², Asn³ -SCT

The 200 ml. of solution from the above synthesis at pH 5.0 wasconcentrated using a SP-25 ion-exchange column. The 25 ml. concentrateremoved from the column with 0.7 molar sodium chloride solution wasdesalted and purified by passing through a Sephadex G-25 (fine)gel-filtration column and eluting with 0.03 molar aqueous acetic acidsolution. The des-Ser², Asn³ -SCT fraction from this column was adjustedto pH 6 by the addition of ammonium hydroxide solution. This solutionwas further purified by ion-exchange chromatography using a WhatmanCM-52 column eluted with ammonium acetate buffer. The des-Ser², Asn³-SCT fraction from this column was adjusted to pH 5.0 by addition ofglacial acetic acid. This solution was concentrated using a SP-SephadexC-25 ion-exchange column. The 30 ml. concentrate removed from the columnwith 0.7 molar sodium chloride solution was desalted with a SephadexG-25 (fine) gel-filtration column. The peptide fraction was collectedand freeze-dried. The product was further purified by partitionchromatography using a Sephadex G-25 fine column and the solvent system:n-butanol, ethanol, 0.2N ammonium acetate containing 0.04% acetic acid(4-1-5). The product elutes from the column at an Rf value of 0.54. Thefractions containing the product were combined and the n-butanol removedby evaporation. The product was recovered by lyophilization. The solidwas then gel-filtered on a Sephadex G-25 (fine) column with 0.2M aceticacid solution. The purified peptide fraction was collected andlyophilized.

The product was obtained as a fluffy white solid. Amino acid analysis ofthe product gave the following ratios with the theoretical values givenin parenthesis: Asp 1.0 (1), Thr 5.2 (5), Ser 2.8 (3), Glu 2.8 (3), Pro2.0 (2), Gly 3.0 (3), Val 0.9 (1), Leu 5.0 (5), His 0.92 (1), Lys 1.9(2), Arg 0.94 (1), Cys 1.91 (2), Tyr 0.91 (1).

Biological Assay Of Calcitonin Analogs In Vivo

The biological potency of des-2-serine, 3-asparagine-salmon calcitoninwas determined by comparing the reduction of serum calcium concentrationwhich followed administration by graded doses of des-Ser², Asn² -SCT andsynthetic salmon calcitonin standard. Rats were divided into four groupsof seven animals, and each group was assigned at random to a dose ofstandard or test solution. Low and high doses were chosen from thelinear portion of the dose-response curve. For the salmon calcitoninstandard, the values were 0.7 and 2.1 ng. peptide/100 g. body weight(BW). This dose approximates 3 and 9 MU/100 g. BW. Peptides were givenby subcutaneous injection (0.2 ml/100 g. BW) and blood was withdrawn 1hour later for serum calcium determination. Sera are processed andanalyzed within two hours of collection. Results were analyzed withintwo hours of collection. Results were analyzed by a 2×2 parallel lineassay [Gaddum, J. H. J. Pharm. Pharmacol., 6, 345 (1953)]. The standardsalmon calcitonin used was independently determined to contain greaterthan 4,000 IU/mg. des-Ser², Asn³ -SCT assayed at 4350 IU/mg.

While only certain embodiments of our invention have been described inspecific detail it will be apparent to those skilled in this art thatmany other specific embodiments may be practiced and many changes may bemade, all within the spirit of the invention and the scope of theappended claims.

What is claimed is:
 1. A peptide having the structure: ##STR26##
 2. Apeptide having the structure: ##STR27##
 3. A peptide having thestructure selected from the group consisting of: ##STR28## where R₁ isS-n-alkyl, Cys or H and R₂ is S-n-alkyl or H, R₁ being S-n-alkyl, Cys orH when R₂ is H and R₂ being S-n-alkyl or H when R₁ is H.
 4. A peptidehaving the structure: ##STR29##
 5. A peptide having the structure:##STR30##